The field of the invention is ornithine δ-aminotransferase (OAT) as a target for anticancer drugs.
The segregation of replicated chromosomes during cell division is executed by the mitotic spindle, a molecular machine assembled transiently for this purpose. The spindle is comprised of filamentous polymerized heterodimeric tubulin radiating from two opposing spindle poles. Tightly regulated microtubule dynamics are necessary for robust bipolar spindle assembly and to ensure fidelity in the separation of sister chromatids at anaphase [1-4]. The small GTPase Ran plays a regulatory role in the initiation of mitotic spindle assembly [5]. At the onset of mitosis, RanGTP is concentrated around condensed chromosomes. Analogous to its role in nucleocytoplasmic transport during interphase, RanGTP stimulates the release of sequestered spindle assembly factors (SAFs) such as NuMA, TPX2, and/or XCTK2 from transport receptors importin β/α and renders them active. These factors, in turn, locally promote microtubule polymerization at spindle poles [6-8].
Diazonamide A (compound 1, Table 1) is a small molecule toxin originally isolated from tissue homogenates of the marine ascidian Diazona angulata [9].
1X = NHR = H(-)-diazonamide A2X = OR = H 3X = O 4X = O
Purified 1 inhibits cultured human cancer cell growth at low nanomolar concentrations. Early analyses at the NCI demonstrated diazonamide toxicity is manifest as an M-phase growth arrest wherein treated cells fail to assemble bipolar mitotic spindles and accumulate in the tetraploid state. COMPARE pattern analysis of observed cytotoxic/cytostatic activities implied a mechanistic correlation between diazonamide and microtubule de-polymerizing agents such as maytansine and vinblastine. Along these lines, 1 was found to inhibit purified tubulin polymerization in vitro as well as dissipate the tubulin cytoskeleton in cells at high concentrations. However, diazonamide A did not compete with vinca alkaloids or nocadazole for tubulin binding [10]. Our own experiments with 1 in BS-C-1 and PTK2 culture showed that, at GI50 concentrations, the molecule had little impact on interphase microtubules—suggesting its catastrophic effect on spindle microtubules was indirect. To test this possibility, a synthetic variant of diazonamide A (2, Table 1) was prepared [11]. Like the natural product, 2 shows potent anti-mitotic activity in cell culture and induces spindle abnormalities during mitosis indistinguishable from those caused by 1. A peripherally biotinylated form of 2 (namely 3, Table 1) also retains this activity (vide infra).
The observation that neither 3 nor a radio-labeled congener of 2 binds specifically to tubulin or microtubules in vitro was the starting point for our studies to determine the functional target of diazonamide A.